Apple beta-galactosidase. Activity against cell wall polysaccharides and characterization of a related cDNA clone.

A beta-galactosidase was purified from cortical tissue of ripe apples (Malus domestica Borkh. cv Granny Smith) using a procedure involving affinity chromatography on lactosyl-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that two polypeptides of 44 and 32 kD were present in the fraction that showed activity against the synthetic substrate p-nitrophenol-beta-D-galactopyranoside. The enzyme preparation was incubated with polysaccharide extracts from apple cell walls containing beta-(1-->4)-linked galactans, and products of digestion were analyzed by gas chromatography. Small amounts of monomeric galactose were released during incubation, showing that the enzyme was active against native substrates. Amino acid sequence information was obtained from the purified protein, and this showed high homology with the anticipated polypeptide coded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldborough, W.R. Woodson [1991] Plant Mol Biol 17: 61-71) and a harvest-related pTIP31 cDNA from asparagus (G. King, personal communication). Using the asparagus cDNA clone as a probe, an apple homolog (pABG1) was isolated. This clone contains a 2637-bp insert, including an open reading frame that codes for a polypeptide of 731 amino acids. Cleavage of an N-terminal signal sequence would leave a predicted polypeptide of 78.5 kD. Genomic DNA analysis and the isolation of other homologous apple clones suggest that pABG1 represents one member of an apple beta-galactosidase gene family. Northern analysis during fruit development and ripening showed accumulation of pABG1-homologous RNA during fruit ripening. Enzyme activity as measured in crude extracts increased during fruit development to a level that was maintained during ripening.     

Structure of ten free N-glycans in ripening tomato fruit. Arabinose is a constituent of a plant N-glycan.

The concentration-dependent stimulatory and inhibitory effect of N-glycans on tomato (Lycopersicon esculentum Mill.) fruit ripening was recently reported (B. Priem and K.C. Gross [1992] Plant Physiol 98: 399-401). We report here the structure of 10 free N-glycans in mature green tomatoes. N-Glycans were purified from fruit pericarp by ethanolic extraction, desalting, concanavalin A-Sepharose chromatography, and amine-bonded silica high performance liquid chromatography. N-Glycan structures were determined using 500 MHz 1H-nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectrometry, and glycosyl linkage methylation analysis by gas chromatography-mass spectrometry. A novel arabinosyl-containing N-glycan, Man alpha 1-->6(Ara alpha 1-->2)Man beta 1-->4GlcNAc beta 1-->4(Fuc alpha 1-->3)GlcNAc, was purified from a retarded concanavalin A fraction. The location of the arabinosyl residue was the same as the xylosyl residue in complex N-glycans. GlcNAc[5']Man3(Xyl)GlcNAc(Fuc)GlcNAc and GlcNAc[5']Man2GlcNAc(Fuc)GlcNAc were also purified from the weakly retained fraction. The oligomannosyl N-glycans Man5GlcNAc, Man6GlcNAc, Man7GlcNAc, and Man8GlcNAc were purified from a strongly retained concanavalin A fraction. The finding of free Man5GlcNAc in situ was important physiologically because previously we had described it as a promoter of tomato ripening when added exogenously. Mature green pericarp tissue contained more than 1 microgram of total free N-glycan/g fresh weight. Changes in N-glycan composition were determined during ripening by comparing glycosyl and glycosyl-linkage composition of oligosaccharidic extracts from fruit at different developmental stages. N-Glycans were present in pericarp tissue at all stages of development. However, the amount increased during ripening, as did the relative amount of xylosyl-containing N-glycans.     

Down-regulation of a ripening-related beta-galactosidase gene (TBG1) in transgenic tomato fruits.

Exo-galactanase/beta-galactosidase (EC 3.2.1.23) activity is thought to be responsible for the loss of galactosyl residues from the cell walls of ripening tomatoes. Transgenic tomato plants (Lycopersicon esculentum Mill cv. Ailsa Craig) with reduced exo-galactanase/beta-galactosidase mRNA were generated to test this hypothesis and to investigate the role of the enzyme in fruit softening. A previously identified tomato beta-galactosidase cDNA clone, TBG1, was used in the experiments. Heterologous expression of the clone in yeast demonstrated that TBG1 could release galactosyl residues from tomato cell wall galactans. Transgenic plants showed a reduction in TBG1 mRNA to 10% of normal levels in the ripening fruits. However, despite the reduction in message, total beta-galactosidase and exo-galactanase activities were unaffected. Furthermore, there was no apparent effect on levels of cell wall galactosyl residues when compared with the control. It was concluded that during the ripening of tomato fruits a family of beta-galactosidases capable of degrading cell wall galactans are active and down-regulation of TBG1 message to 10% was insufficient to alter the degree of galactan degradation.     

Biochemical basis of high-temperature inhibition of ethylene biosynthesis in ripening tomato fruits

Biggs, M. S., Woodson, W. R. and Handa, A. K. 1988. Biochemical basis of high-temperature inhibition of ethylene biosynthesis in ripening tomato fruits. Physiol. Plant. 72: 572578
Incubation of fruits of tomato ( Lycopersicon esculentum Mill. cv. Rutgers) at 34°C or above resulted in a marked decrease in ripening-associated ethylene production. High temperature inhibition of ethylene biosynthesis was not associated with permanent tissue damage, since ethylene production recovered following transfer of fruits to a permissive temperature. Determination of pericarp enzyme activities involved in ethylene biosynthesis following transfer of fruits from 25°C to 35 or 40°C revealed that 1-aminocyclopropane-l-carboxylic acid (ACC) synthase (EC 4.4.1.14) activity declined rapidly while ethylene forming enzyme (EFE) activity declined slowly. Removal of high temperature stress resulted in more rapid recovery of ACC synthase activity relative to EFE activity. Levels of ACC in pericarp tissue reflected the activity of ACC synthase before, during, and after heat stress. Recovery of ethylene production following transfer of pericarp discs from high to permissive temperature was inhibited in the presence of cycloheximide, indicating the necessity for protein synthesis. Ethylene production by wounded tomato pericarp tissue was not as inhibited by high temperature as ripening-associated ethylene production by whole fruits.     

Ectopic expression of apple fruit homogentisate phytyltransferase gene (MdHPT1) increases tocopherol in transgenic tomato (Solanum lycopersicum cv. Micro-Tom) leaves and fruits

Homogentisate phytyltransferase (HPT) is an important enzyme in the biosynthesis of tocopherols (vitamin E). Herein, an HPT homolog (MdHPT1) was isolated from apple (Malus domestica Borkh. cv. Fuji) fruits, whose gene expression level gradually decreased during fruit ripening, reaching a background level in ripened apple fruits. The amounts of α- and γ-tocopherols, two major tocopherols in plant organs, were 5- to 14-fold lower in the fruits than in the leaves and flowers of apple plants. Transgenic tomato plants (Solanum lycopersicum cv. Micro-Tom) overexpressing MdHPT1 were next constructed. Transgenic independent T(1) leaves contained ～1.8- to 3.6-fold and ～1.6- to 2.9-fold higher levels of α-tocopherol and γ-tocopherol, respectively, than those in control plants. In addition, the levels of α-tocopherol and γ-tocopherol in 35S:MdHPT1 T(1) fruits increased up to 1.7-fold and 3.1-fold, respectively, as compared to the control fruits, indicating that an increase in α-tocopherol in fruits (maximal 1.7-fold) was less evident than that in leaves (maximal 3.6-fold). This finding suggests that the apple MdHPT1 plays a role in tocopherol production in transgenic tomatoes.     

Effect of Heat Treatment on the Development of Polygalacturonase Activity in Tomato Fruit during Ripening

When mature green tomato fruits are stored at 22?C for 30 days,they ripen normally and soften, but if they are kept at 33?Cfor 15 days (heat treatment), then stored at 22?C they do notsoften. The effect of heat treatment on the development of polygalacturonase(PG, EC 3.2.1.15 [EC] ) activities in tomato fruits during storagetherefore was studied. When mature green tomato fruits werestored at 22?C, PG activity, which had not been detectable inthe fruits, appeared as the color changed and increased dramaticallythereafter. PG activity, however, did not appear during heattreatment. When heat-treated fruits were transferred to 22?C,PG activity appeared after a 6-day lag period and increasedduring the next 30 days at 22?C to about 15% of the value detectedin ripe tomato fruits. The PG in ripe tomato fruits was composed of two isoenzymesthat had different mol wts. A high molecular form (PG-1, molwt 100K) appeared during the early stage of ripening and a lowmolecular form (PG-2, mol wt 44K) a little later. PG-2 increasedvigorously during ripening and eventually accounted for mostof the enzyme activity in the ripe fruits. Only a single isoenzyme(Y-PG, mol wt 100K), however, was detected in heat-treated tomatofruits stored at 22?C for 30 days. PG-1 and Y-PG gave the samemol wt on Sephacryl S-200 gel nitration, but could be separatedby Toyopearl HW-55 F gel filtration. (Received October 31, 1983; Accepted February 20, 1984)     

Pepper beta-galactosidase 1 (PBG1) plays a significant role in fruit ripening in bell pepper (Capsicum annuum)

During bell pepper (Capsicum annuum L.) fruit ripening, beta-galactosidase activity increased markedly as compared with other glycosidases. We purified 77.5 kDa exo-1,4-beta-D-galactanase from red bell pepper fruit classified as beta-galactosidase II. A marked decrease in galactose content appeared during fruit ripening, especially in the pectic fraction. The purified enzyme hydrolyzed a considerable amount of galactose residues in this fraction. We isolated bell pepper beta-galactosidase (PBG1) cDNA. This PBG1 protein contained the putative active site, G-G-P-[LIVM]-x-Q-x-E-N-E-[FY], belonging to glycosyl hydrolase family 35. Quantitative RT-PCR revealed that the expression of PBG1 in red fruit was significantly stronger than that from any other tissues. Moreover, expression of PBG1 occurred prior to that of pepper endo-polygalacturonase 1 (PPG1), the major fruit-ripening enzyme. Based on these results, it appears that the hydrolysis of galactose residues in pectic substances is the first event in the ripening process in bell pepper fruit.     

Purification and characterization of a novel enzyme, alpha-neoagarooligosaccharide hydrolase (alpha-NAOS hydrolase), from a marine bacterium, Vibrio sp. strain JT0107.

A novel enzyme, alpha-neoagarooligosaccharide hydrolase (EC 3.2.1.-), which hydrolyzes the alpha-1,3 linkage of neoagarooligosaccharides to yield agaropentaose (O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose], agarotriose [O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro- alpha-L-galactopyranosyl (1-->3)-D-galactose], agarobiose [O-beta-D-galactopyranosyl(1-->4)-3,6-anhydro-L-galactose], 3,6-anhydro-L-galactose, and D-galactose was isolated from the marine bacterium Vibrio sp. strain JT0107 and characterized. This enzyme was purified 383-fold from cultured cells by using a combination of ammonium sulfate precipitation, successive anion-exchange column chromatography, gel filtration, and hydroxyapatite chromatography, gel filtration, and hydroxyapatite chromatography. The purified protein gave a single band (M(r), 42,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the M(r) by the gel filtration method gave a value of 84,000, indicating that the enzyme is dimeric. Amino acid sequence analysis revealed it to have a single N-terminal sequence that has no sequence homology to any other known agarases. The optimum temperature and pH were 30 degrees C and 7.7, respectively. The Km and maximum rate of metabolism for neoagarobiose were 5.37 mM and 92 U/mg of protein, respectively.     

Endopolygalacturonase in Apples (Malus domestica) and Its Expression during Fruit Ripening

The activity of polygalacturonase (PG) has been detected in ripe McIntosh apples (Malus domestica Borkh. cv McIntosh) both by enzyme activity measurement and immunoblotting using an anti-tomato-PG antibody preparation. PG activity increased during fruit ripening and remained steady, or decreased slightly, after 5 months of controlled atmospheric storage. The enzyme had a relative molecular weight of 45,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 56,000 to 61,000 when determined by gel filtration. Viscosity and reducing end group measurements with a commercial pectin preparation showed that the enzyme is endo acting. In RNA and DNA blot hybridization experiments, a full-length tomato PG cDNA hybridized with the apple RNA and DNA, showing the identity of genes encoding the activity of the enzyme in tomato and apple.     

Delay of tomato fruit ripening by an oligosaccharide N-glycan. Interactions with IAA, galactose and lectins

The unconjugated N-glycans Manα1→6(Manα→3)Manα1→6(Manα1→3)-Manβ1→4GlcNAc (Man₅GlcNAc) and Manα1→6(Manα1→3)(Xy1β1→2)-Manβ1→4GlcNAcβ1→4(Fucα1→3)GlcNAc were shown to stimulate and delay ripening of mature green tomato fruit (Lycopersicon esculentum Mill. cv. Rutgers) at 1 and 10 ng (g fresh weight)⁻¹, respectively (Priem and Gross 1992, Plant Physiol. 98: 399–401). Also, the occurrence and structure of 10 unconjugated N-glycans, including Man₅GlcNAc, in tomato fruit were recently reported (Priem et al. 1993, Plant Physiol. 102: 445–458). In this work, we studied the potential interaction between Man₅GlcNAc and several compounds by using a system that allowed pericarp discs to be kept up to 14 days after excision. Studies were performed to determine the effect of Man₅GlcNAc, indole-3-acetic acid (IAA), concanavalin A and tomato lectin on ripening as defined by red coloration of the skin. Ripening in pericarp discs, unlike that in intact fruit, was unaffected by 1 ng (g fresh weight)⁻¹ Man₅GlcNAc. However, discs showed delayed ripening with 10 ng (g fresh weight)⁻¹, and the delay of coloration was galactose dependent. Man₅GlcNAc at 10 ng (g fresh weight)⁻¹ inhibited the stimulation of coloration induced by 100 μ IAA in the presence of 40 μg (g fresh weight)⁻¹ galactose. Man₅GlcNAc lost biological activity in the presence of the two lectins. These results support the possible interaction of IAA and various oligosaccharides during plant growth and development, and for the first time suggest a physiological significance for tomato lectin.     

Solution 1H nuclear magnetic resonance determination of the distal pocket structure of cyanomet complexes of genetically engineered sperm whale myoglobin His64 (E7)-->Val, Thr67 (E10)-->Arg. The role of distal hydrogen bonding by Arg67 (E10) in modulating ligand tilt.

Sequence-specific 2D methodology has been used to assign the 1H NMR signals for all active site residues in the paramagnetic cyano-met complexes of sperm whale synthetic double mutant His64[E7]-->Val/Thr67[E10]-->Arg (VR-met-MbCN) and triple mutant His64[E7]-->Val/Thr67[E10]-->Arg/Arg45[CD3]-->Asn (VRN-metMbCN). The resulting dipolar shifts for noncoordinated proximal side residues were used to quantitatively determine the orientation of the paramagnetic susceptibility tensor in the molecular framework for the two mutants, which were found indistinguishable but distinct from those of both wild-type and the His64[E7]-->Val single point mutant (V-metMbCN). The observed dipolar shifts for the E helix backbone protons and Phe43[CD1], together with steady-state nuclear Overhauser effect between the E helix and the heme, were analyzed to show that both the E helix and Phe43[CD1] move slightly closer to the iron to minimize the vacancy resulting from the His64[E7]-->Val substitution, as found in V-metMbCN (Rajarathnam, K., J. Qin, G.N. LaMar, M. L. Chiu, and S. G. Sligar. 1993. Biochemistry. 32:5670-5680). The dipolar shifts of the mutated Val64[E7] and Arg67[E10] allow the determination of their orientations relative to the heme, and the latter residue is shown to insert into the pocket and provide a hydrogen bond to the coordinated ligand, as found in the naturally occurring ValE7/ArgE10 genetic variant, Aplysia limacina Mb. The oxy-complex of both A. limacina Mb and VR-Mb, VRN-Mb have been proposed to be stabilized by this hydrogen bonding interaction (Travaglini Allocatelli, C. et al. 1993. Biochemistry. 32:6041-6049). The magnitude of the tilt of the major magnetic axes from the heme normal in VR-metMbCN and VRN-metMbCN, which is related to the tilt of the ligand, is the same as in wild-type or V-metMbCN, but the direction of tilt is altered from that in V-metMbCN. It is concluded that the change in the direction of the ligand tilt in both the double and triple mutants, as compared to WT metMbCN and V-metMbCN single mutant, is due to the attractive hydrogen-bonding between ArgE10 and the bound cyanide.     

Effect of the Colorless non-ripening mutation on cell wall biochemistry and gene expression during tomato fruit development and ripening

The Colorless non-ripening (Cnr) mutation in tomato (Solanum lycopersicum) results in mature fruits with colorless pericarp tissue showing an excessive loss of cell adhesion (A.J. Thompson, M. Tor, C.S. Barry, J. Vrebalov, C. Orfila, M.C. Jarvis, J.J. Giovannoni, D. Grierson, G.B. Seymour [1999] Plant Physiol 120: 383-390). This pleiotropic mutation is an important tool for investigating the biochemical and molecular basis of cell separation during ripening. This study reports on the changes in enzyme activity associated with cell wall disassembly in Cnr and the effect of the mutation on the program of ripening-related gene expression. Real-time PCR and biochemical analysis demonstrated that the expression and activity of a range of cell wall-degrading enzymes was altered in Cnr during both development and ripening. These enzymes included polygalacturonase, pectinesterase (PE), galactanase, and xyloglucan endotransglycosylase. In the case of PE, the protein product of the ripening-related isoform PE2 was not detected in the mutant. In contrast with wild type, Cnr fruits were rich in basic chitinase and peroxidase activity. A microarray and differential screen were used to profile the pattern of gene expression in wild-type and Cnr fruits. They revealed a picture of the gene expression in the mutant that was largely consistent with the real-time PCR and biochemical experiments. Additionally, these experiments demonstrated that the Cnr mutation had a profound effect on many aspects of ripening-related gene expression. This included a severe reduction in the expression of ripening-related genes in mature fruits and indications of premature expression of some of these genes in immature fruits. The program of gene expression in Cnr resembles to some degree that found in dehiscence or abscission zones. We speculate that there is a link between events controlling cell separation in tomato, a fleshy fruit, and those involved in the formation of dehiscence zones in dry fruits.     

Molecular analysis of Hurler syndrome in Druze and Muslim Arab patients in Israel: multiple allelic mutations of the IDUA gene in a small geographic area.

The mutations underlying Hurler syndrome (mucopolysaccharidosis IH) in Druze and Muslim Israeli Arab patients have been characterized. Four alleles were identified, using a combination of (a) PCR amplification of reverse-transcribed RNA or genomic DNA segments, (b) cycle sequencing of PCR products, and (c) restriction-enzyme analysis. One allele has two amino acid substitutions, Gly409-->Arg in exon 9 and Ter-->Cys in exon 14. The other three alleles have mutations in exon 2 (Tyr64-->Ter), exon 7 (Gln310-->Ter), or exon 8 (Thr366-->Pro). Transfection of mutagenized cDNAs into Cos-1 cells showed that two missense mutations, Thr366-->Pro and Ter-->Cys, permitted the expression of only trace amounts of alpha-L-iduronidase activity, whereas Gly409-->Arg permitted the expression of 60% as much enzyme as did the normal cDNA. The nonsense mutations were associated with abnormalities of RNA processing: (1) both a very low level of mRNA and skipping of exon 2 for Tyr64-->Ter and (2) utilization of a cryptic splice site for Gln310-->Ter. In all instances, the probands were found homozygous, and the parents heterozygous, for the mutant alleles, as anticipated from the consanguinity in each family. The two-mutation allele was identified in a family from Gaza; the other three alleles were found in seven families, five of them Druze, residing in a very small area of northern Israel. Since such clustering suggests a classic founder effect, the presence of three mutant alleles of the IDUA gene was unexpected.     

RFLP analysis of 1-aminocyclopropane-1-carboxylate synthase ACC2 and ACC4 genes from Polish cultivars of tomato

An important trait of tomato is the rate of fruit ripening, strongly dependent on ethylene production. The ripening-related ethylene synthesis in tomato is controlled mainly by 1-aminocyclopropane-1-carboxylate synthase LE-ACS2 and LE-ACS4 isoenzymes (Rottmann et al., 1991, J. Mol. Biol. 222: 937; Lincoln et al., 1993, J. Biol. Chem. 268: 19422; Barry et al., 2000, Plant Physiol. 123: 979). In spite of numerous reports on the LE-ACS2 and LE-ACS4 gene expression, only ones considered the genomic organisation each of these genes (Rottmann et al., 1991; Lincoln et al., 1993) reported one copy of each of these genes in tomato cv VF36. In this article we suggest that the genomic organisation of LE-ACS2 and LE-ACSS4 genes may depend on tomato cultivars and may differ from that described by the above authors. The results of Southern analyses of genomic DNAs from 17-day old seedlings (cultivars Jaga, Halicz, Betalux, New Yorker) imply that the genomic organisation of LE-ACS2 and LE-ACS4 genes in Polish cultivars differs from that reported for cv VF36.     

Purification and some properties of alpha-L-arabinofuranosidase from Bacillus subtilis 3-6.

alpha-L-Arabinofuranosidase (EC 3.2.1.55) was purified from culture supernatant of Bacillus subtilis 3-6. The enzyme had a molecular weight of 61,000 and displayed maximum activity at pH 7.0 and 60 degrees C. It released arabinose from O-alpha-L-arabinofuranosyl-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-x ylopyranos e (A1X2), O-beta-D-xylopyranosyl-(1-->4)-[O-alpha-L-arabinofuranosyl-(1-->3)]- O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (A1X3), and arabinan, but not from O-beta-D-xylopyranosyl-(1-->2)-O-alpha-L- arabinofuranosyl-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-O-beta-D-xylopyr anosyl- (1-->4)-D-xylopyranose (A1X4), arabinoxylan, gum arabic, or arabinogalactan.     

Purification and characterization of a beta-galactosidase from peach (Prunus persica)

A beta-galactosidase (EC 3.2.1.23) from peach (Prunus persica cv Mibackdo) was purified and characterized. The purified peach beta-galactosidase was 42 kDa in molecular mass and showed high enzyme activity against a the beta-galactosidase substrate, rho-nitrophenyl-beta-D-galactopyranoside. The Km and Vmax values of the enzyme activity of the peach beta-galactosidase were 5.16 and 0.19 mM for rho-nitrophenyl-beta-D-galactopyranoside mM/h, respectively. The optimum pH of the enzyme activity was pH 3.0, but it was relatively stable from pH 3.0-10.0. The temperature optimum was 50 degrees C. The enzyme activities were not improved in the buffers that contained Ca2+, Cu2+, Zn2+, and Mg2+, which indicates that the purified peach beta-galactosidase did not require these cations as co-factors. However, the enzyme was completely inhibited by Hg2+. The purified protein was cross-reacted with an antibody against the persimmon fruit beta-galactosidase. A further comparison of the N-terminal amino acid sequence of the purified protein showed high homologies to those of beta-galactosidase in apple (87%), persimmon (80%), and tomato (87%). Therefore, enzymatic, immunological, and molecular evidences in this study indicate that the purified 42-kDa protein is a peach beta-galactosidase.     

Structural studies of lipooligosaccharides from Haemophilus ducreyi ITM 5535, ITM 3147, and a fresh clinical isolate, ACY1: evidence for intrastrain heterogeneity with the production of mutually exclusive sialylated or elongated glycoforms.

The structures of the lipooligosaccharides (LOSs) from Haemophilus ducreyi ITM 5535 and ITM 3147 and a fresh clinical isolate, ACY1, have been investigated. Oligosaccharides were obtained from phenol-water-extracted LOS by mild acid hydrolysis and were studied by methylation analysis, fast atom bombardment and electrospray ionization mass spectrometry, and nuclear magnetic resonance spectroscopy. The major oligosaccharide obtained from all strains was a nonasaccharide with the structure beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-D-Galp-(1-->4)-D-a lpha-D-Hepp- (1-->6)-beta-D-Glcp-(1-->[L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp - (1-->3)]4)-L-alpha-D-Hepp-Kdo (Kdo stands for 3-deoxy-D-manno-octulosonic acid) and is thus identical to that identified as the major oligosaccharide in H. ducreyi ITM 2665 (E. K. H. Schweda, A. C. Sundström, L. M. Eriksson, J.A. Jonasson, and A. A. Lindberg, J. Biol. Chem. 269:12040-12048, 1994). Electrospray ionization mass spectrometry on O-deacylated LOS from H. ducreyi ITM 5535 obtained after treatment with anhydrous hydrazine gave evidence for the presence of a sialylated major compound, Neu5Ac alpha(2-->3)-beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-D-Gal p- (1-->4)-D-alpha-D-Hepp-(1-->6)-beta-D-Glcp-(1-->[L-alpha-D-Hepp -(1-->2)-L- alpha-D-Hepp-(1-->3)]4)-L-alpha-D-Hepp-Kdo(P)-O-deacylated lipid A (Neu5Ac stands for N-acetylneuraminic acid). However, an even larger oligosaccharide could be isolated from all strains as a minor component, viz., the undecasaccharide beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-d-Galp-(1-->4)-beta-D-glcNAcp-(1-->3)-beta-D-Galp-(1-->4)-D-alpha-D-Hepp-(1-->6)-beta-D-Glcp-(1-->[L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp-(1-->3)]4-L-alpha-D-Hepp-Kdo, which represents an N-acetyl lactosamine disaccharide unit elongation of the LOS outer core. No Sialylation of this latter minor component undecasaccharide was detected.     

Measurement of beta(1-->3)-glucans in occupational and home environments with an inhibition enzyme immunoassay.

beta (1-->3)-Glucans are known for their potent ability to induce nonspecific inflammatory reactions and are believed to play a role in bioaerosol-induced respiratory symptoms. An inhibition enzyme immunoassay (EIA) was developed for the quantitation of beta (1-->3)-glucans in dust samples from occupational and residential environments. Immunospecific rabbit antibodies were produced by immunization with bovine serum albumin-conjugated laminarin [beta (1-->3)-glucan] and affinity chromatography on epoxy-Sepharose-coupled beta (1-->3)-glucans. The laminarin-based calibration curve in the inhibition EIA ranged from approximately 40 to 3,000 ng/ml (15 to 85% inhibition). Another beta (1-->3)-glucan (curdlan) showed a similar inhibition curve but was three to five times less reactive on a weight basis. Pustulan, presumed to be a beta (1-->6)-glucan, showed a parallel dose-response curve at concentrations 10 times higher than that of laminarin. Control experiments with NaIO4 and beta (1-->3)-glucanase treatment to destroy beta (1-->6)- and beta (1-->3)-glucan structures, respectively, indicate that the immunoreactivity of pustulan in the assay was due to beta (1-->3)-glucan and not to beta (1-->6)-glucan structures. Other polysaccharides, such as mannan and alpha (1-->6)-glucan, did not react in the inhibition EIA. Beta (1-->3)-Glucan extraction of dust samples in water (with mild detergent) was performed by heat treatment (120 degrees C) because aqueous extracts obtained at room temperature did not contain detectable beta (1-->3)-glucan levels. The assay was shown to detect heat-extractable beta (1-->3)-glucan in dust samples collected in a variety of occupational and environmental settings. On the basis of duplicate analyses of dust samples, a coefficient of variation of approximately 25% was calculated. It was concluded that the new inhibition EIA offers a useful method for indoor beta (1-->3)-glucan exposure assessment.     

Functional role of proline and tryptophan residues highly conserved among G protein-coupled receptors studied by mutational analysis of the m3 muscarinic receptor.

Most G protein-coupled receptors contain a series of highly conserved proline and tryptophan residues within their hydrophobic transmembrane domains (TMD I-VII). To study their potential role in ligand binding and receptor function, the rat m3 muscarinic acetylcholine receptor was used as a model system. A series of mutant receptors in which the conserved proline and tryptophan residues were individually replaced with alanine and phenylalanine, respectively, was created and transiently expressed in COS-7 cells. [3H]N-methylscopolamine ([3H]NMS) saturation binding studies showed that three of the seven mutant receptors studied (Pro242-->Ala, TMD V; Pro505-->Ala, TMD VI; Pro540-->Ala, TMD VII) were expressed at 35-100 times lower levels than the wild-type receptor while displaying 'm3-like' antagonist binding affinities. Pro201-->Ala (TMD IV) showed drastically reduced binding affinities (up to 450-fold) for both muscarinic agonists and antagonists. Whereas most mutant receptors retained strong functional activity, Pro540-->Ala (TMD VII) was found to be severely impaired in its ability to stimulate carbachol-induced phosphatidyl inositol hydrolysis (Emax approximately 25% of wild type m3). Interestingly, this mutant receptor bound muscarinic agonists with 7- to 19-fold higher affinities than the wild type receptor. The Trp-->Phe substitutions (Trp192-->Phe, TMD IV; Trp503-->Phe, TMD VI; Trp530-->Phe, TMD VII) resulted in less pronounced changes (compared with the Pro-->Ala mutant receptors) in both ligand binding and receptor function. Our data indicate that the proline residues that are highly conserved across the entire superfamily of G protein-coupled receptors play key roles in receptor expression, ligand binding and receptor activation.